Project Summary/Abstract Protein therapeutics are the fastest growing segment of the pharmaceutical market, yet there are two important challenges associated with the discovery and development of biologic drugs: epitope/paratope mapping and 3D structural analysis. Epitope and paratope mapping are essential parts of identifying the most therapeutically relevant lead candidates for advancement to in vivo testing. Accurately mapping epitopes can help identify therapeutic candidates that bind to the desired region of an antigen based on the mechanism of action, while characterizing paratopes frequently forms the basis for intellectual property strategies. Protein 3D structural analysis is crucial for many downstream activities throughout the drug development process, and it most often is required to ensure that manufacturing, shipping, storage, and formulation do not have an unintended impact on protein structure. In addition, 3D structural analysis plays a critical role in ensuring biosimilars under development are structurally similar to their branded originator drug. Current analytical strategies for epitope/paratope mapping and 3D structural analysis of protein therapeutics are often inadequate for the early stages of drug discovery and development. In response, QuarryBio and its research partners at Indiana University and the University of Massachusetts Amherst have developed a new mass spectrometry-based method that uses covalent labeling to identify epitopes and paratopes and can detect changes in protein 3D structure accurately and sensitively. Research during Phase I demonstrated that this method is quick, easy to use and provides sufficient resolution for both epitope mapping and 3D structural analysis of protein therapeutics. Success during Phase I was possible because of the creation of a customized data analysis software pipeline that could successfully identify and quantify covalently labeled sites in proteins, allowing the needed protein structural information to be obtained. Phase II will build off these successful preliminary studies, thereby preparing this technology for commercialization. Three specific aims will be pursued: 1) we will demonstrate the ability of our covalent labeling technology to provide robust structural information and to map epitopes and paratopes in mAb/antigen complexes; 2) we will automate data analysis and visualization by creating protocols that minimize the manual manipulations associated with our customized software; and 3) we will develop laboratory automation for sample preparation activities to improve the reproducibility of our measurements. At the end of Phase II, we expect that our covalent labeling technology and data analysis software pipeline will be fully ready for commercialization. QuarryBio has fee-for-service contracts in place with seven biotech clients, demonstrating the commercial potential of this technology. The work proposed here will significantly improve the quality, reproducibility, and throughput of our approach and demonstrate how our methodology can be used for key decision steps during early stage biologic drug discovery and development.